1. Field of the Invention
The present invention relates to a method of amplifying/expanding hematopoietic stem cells. In particular, the present invention relates to the amplification/expansion of human bone marrow stem cells by culturing cells with endothelial cells in the presence of growth factors or cytokines.
2. Description of the Prior Art
Hematopoiesis, the formation of mature blood cells, involves a complex scheme of multilineage differentiation (Metcalf, Nature 339:27-30, 1989). Hematopoiesis occurs mainly in the bone marrow where hematopoietic stem cells (pluripotential stem cells) proliferate and differentiate into progenitor cells which then develop into different types of mature blood cells (Gordon et al., Bone Marrow Transplant 4:335, 1989; Dexter et al., Ann. Rev. Cell Bio. 3:423, 1987). The hematopoietic stem and progenitor cell is functionally characterized by its extensive and prolonged self-renewal capacity as well as its ability to differentiate and thereby give rise to cells of all lymphohematopoietic lineages (Sprangruide et al., Science 241:58, 1988; Terstappen et al., Blood 77:1218, 1991; Civin et al., Exp. Hematol. 15:10, 1987; Civin et al., J. Immunol. 133:157, 1984; Strauss et al., Exp. Hematol. 14:878, 1986). Phenotypically, the only well defined human hematopoietic stem and progenitor cell marker at present is the CD34 hematopoietic cell surface antigen (Civin et al., J. Immunol. 133:157, 1984; Strauss et al., Exp. Hematol. 14:878, 1986). This cell surface antigen is a highly glycosylated 115-Kd type I integral membrane protein of unknown function (Civin, Exp. Hematol. 18:461, 1990). The sequence of the human CD34 cDNA suggests the presence of several O-linked glycosylation sites (Simmons et al., J. Immunol. 148:267, 1992) and that the attachment of lineage-specific glycans to the CD34 backbone may permit binding to lectins on marrow stromal cells or the extracellular matrix. The CD34 antigen is expressed by approximately 1-5% of the human bone marrow cell population (Civin et al., Exp. Hematol. 15:10, 1987; Civin et al., J. Immunol. 133:157, 1984; Strauss et al., Exp. Hematol. 14:878, 1986) and include pluripotent as well as precursors for each of the hematopoietic cell lineages (Andrews et al., J. Exp. Med. 172:355, 1990; Berstein et al., Blood 77:2316, 1991). In addition, the CD34 antigen is expressed on human vascular endothelial cells (Fina et al., Blood 75:2417, 1990), suggesting a possible role for the antigen in adhesion or cellular interactions. Purified CD34.sup.+ stem and progenitor cells can reconstitute hematopoiesis in vivo (Berenson et al., J. Clin. Invest. 81:951, 1988; Berenson, et al., Blood 77:1717, 1991) and support myelopoiesis for several months in association with stromal cells in long-term bone marrow cultures (Allan et al, Exp. Hematol. 12:517, 1984; Andrews et al, J. Exp. Med. 172:355, 1990; Sutherland et al, Blood 74:1563, 1989; Gordon et al, J. Cell Physiol. 130:150, 1987; Gordon et al, Br. J. Haematol. 60:129, 1985; Verfaillie et al, J. Exp. Med. 172:509, 1990). Additional studies have demonstrated that the pluripotent hematopoietic stem cell can be identified by additional phenotypic markers, singly and in combination. The most primitive pluripotent human bone marrow hematopoietic stem cells are small (low forward light scatter and side scatter) CD34.sup.+, Thy1.sup.+/-, c-kit.sup.+, HLA-DR.sup.-, CD38.sup.-, CD15.sup.-, rhodamine-123 dull and 4-hydroperoxycyclophosphamide-resistant cells, but are hematopoietic lineage marker negative (Lin.sup.-) (Baum et al, Proc Natl Acad Sci USA 89:2804, 1992; Briddle et al, Blood 79:3159, 1992; Craig et al, J Exp Med 177:1331, 1993). Similarly, recent purification experiments have shown that the most primitive murine hematopoietic stem cells have been isolated with the use of a variety of phenotypic markers, such as Thy-1, c-kit, wheat-germ agglutinin (WGA), and stem cell antigen (Okada et al, Blood 78:1706, 1991; Ikuta and Weissman, Proc Natl Acad Sci USA 89:1502, 1992) but are Lin.sup.- (Sprangrude et al, Science 241:58, 1988).
The bone marrow serves in vivo as the requisite microenvironment where constitutive hematopoiesis, stem cell differentiation and stem cell self-renewal occurs (Gordon et al., Bone Marrow Transplant 4:335, 1989; Dexter et al., Ann. Rev. Cell Bio. 3:423, 1987; Allan et al., Exp. Hematol. 12:517, 1984). This microenvironment has two major components--the lymphohematopoietic elements and the bone marrow stroma. The bone marrow stroma, made up of fibroblasts, endothelial cells, adipocytes and macrophages/monocytes, provides a heterogeneous adherent cell layer. Only these heterogeneous adherent cell layers have been shown to be effective in supporting long-term in vitro CD34.sup.+ stem and progenitor cell proliferation and differentiation (Dorshkind, Annu. Rev. Immunol. 8:111, 1990; Dexter et al., J. Cell Physiol. 91:335, 1977; Allan et al., Exp. Hematol. 12:517, 1984). Within the bone marrow stroma, CD34.sup.+ hematopoietic stem and progenitor cells undergo self-renewal, proliferation and differentiation (Andrews et al., J. Exp. Med. 172:355, 1990; Sutherland et al., Blood 74:1563, 1989; Gordon et al., J. Cell Physiol. 130:150,1987; Gordon et al., Br. J. Haematol. 60:129, 1985). The proliferation and differentiation of CD34.sup.+ stem and progenitor cell in stromal dependent cultures is thought to involve cell-to-cell interactions (Andrews et al., J. Exp. Med. 172:355, 1990; Verfaillie et al., J. Exp. Med, 172:509, 1990; Gordon et al., J. Cell Physiol. 130:150, 1987), stroma derived cytokines (Berstein et al., Blood 77:2316, 1991; Dorshkind, Annu. Rev. Immunol. 8:111, 1990; Dexter et al., J. Cell Physiol. 91:335, 1977; Clark et al., Science 236:1229, 1987) and extracellular matrix proteins (Campbell et al., J. Clin. Invest. 75:2085, 1985; Campbell et al., Nature 329:744, 1987; Tsai et al., Blood 69:1587, 1987; Liesveld et al., Blood 73:1794, 1989).
There has been much interest and work in establishing an in vitro culture system for CD34.sup.+ hematopoietic stem and progenitor cells (Edgington et al., Bio/Technology 10:1099, 1992). CD34.sup.+ hematopoietic stem and progenitor cells cultured in vitro could be used in human therapeutics. For example, such cells could be employed in bone marrow transplantation (BMT). Most BMT protocols attempt to restore hematopoietic function following exposure to myeloablative agents. BMT is, therefore, an important adjunct to the therapeutic treatment of advanced malignancies (both hematologic and non-hematologic), intrinsic marrow defects, and bone marrow injury by extrinsic agents (for example, radiation or toxins). In addition, BMT in combination with developing genetic therapy technology is expected to play an increasing role in the treatment of numerous diseases. For a general review, see Negrin et al. (Marrow Transplantation Reviews 2:23, 1992) and Antman (Marrow Transplantation Reviews 2:27, 1992).
Hematopoietic culture systems can be broadly classified into two groups, liquid culture and stromal coculture systems. Liquid culture systems grow CD34.sup.+ hematopoietic stem and progenitor cells suspended in liquid media with additional growth factors and cytokines (Haylock et al, Blood 80: 1405, 1992; Brugger et al, Blood 81: 2579, 1993). Although liquid cultures are easy to maintain and are technically well suited for large scale expansion of CD34.sup.+ hematopoietic stem and progenitor cells for use in therapy, they have uniformly been unsuccessful in generating expanded numbers of CD34.sup.+ stem and progenitor cells that are necessary for long term engraftment. This is also reflected in the fact that it is difficult to maintain these cultures over a long period of time (months) as the CD34.sup.+ stem cells all quickly differentiate into more mature cells. The inability to maintain and expand a proliferating pool of undifferentiated CD34.sup.+ stem and progenitor cells is thought to be due to the lack of the appropriate microenvironment generated by the bone marrow stromal elements. To rectify this problem, stromal coculture systems grow CD34.sup.+ hematopoietic stem and progenitor cells on or within an adherent layer of bone marrow stroma (a heterogeneous population of endothelial cells, adipocytes, fibroblasts and macrophages), with or without the addition of growth factors and cytokines.
One of the first long-term bone marrow culture systems (LTBMCS) was described by Dexter et al., (Dexter et al., Ann. Rev. Cell Bio. 3:423, 1987). Dexter et al.'s LTBMCS demonstrated that sustained cellular growth and development can be accomplished under in vitro culture conditions. In this system, an adherent bone marrow stromal layer appears to provide the growth factors and cellular environment necessary for the proliferation of CD34.sup.+ hematopoietic stem and progenitor cells and their differentiation into a variety of committed progenitor cells (Andrews et al., J. Exp. Med. 172:355, 1990; Gordon et al., Br. J. Haematol. 60:129, 1985). However, the microenvironmental influences and regulation of hematopoiesis in this culture system are difficult to analyze due to the heterogeneity of the cell types that make up the bone marrow stroma. This complexity of the microenvironment also greatly hinders attempts to define elements that are responsible for specific stages of hematopoietic cell development.
Despite the ability of LTBMCS to generate a sustained output of cells over a long period (indicating the maintenance of a CD34.sup.+ stem and progenitor cell pool), these systems are presently difficult to utilize in a therapeutic setting. First, there is the aforementioned complexity of the heterogeneous cellular microenvironment which confounds careful analysis of the ongoing biological mechanisms and prevents substantial improvement of the culture system. Second, LTBMCS require the establishment of a bone marrow stromal layer prior to the seeding of hematopoietic cells into the culture. Establishment of the stromal layer often takes weeks, a significant delay. This also hinders large scale LTBMCS which will be required for therapeutic applications. Finally, there is not a rapid and significant output of CD34.sup.+ hematopoietic stem and progenitor cells from these culture systems, again limiting their usefulness in the therapeutic setting.
In an effort to overcome the complexity and the limitations of the Dexter LTBMCS, perfusion bioreactor systems were developed in which CD34.sup.+ hematopoietic stem and progenitor cells could be cultured. Perfusion bioreactors help to maintain a defined culture environment and reduce physical disruption. See, for example, Schwartz et al. (PNAS USA 88:6760, 1991), Koller et al. (Bio/Technology 11:358, 1993), Koller et al. (Blood 82:378, 1993), and Palsson et al. (Bio/Technology 11:368, 1993). However, most of these perfusion bioreactor systems are modified liquid culture systems and suffer many of the same shortcomings in their inability to amplify/expand the pluripotent hematopoietic CD34.sup.+ stem and progenitor cell pool, cells essential for long term/permanent engraftment and reconstitution of the hematopoietic system. This limits the usefulness of these culture systems for bone marrow transplantation and gene therapy of CD34.sup.+ hematopoietic stem and progenitor cells.
Thus, the need for in vitro culture systems capable of clinically useful amplification of human CD34.sup.+ hematopoietic stem and progenitor cells as well as other hematopoietic elements continues. A system capable of producing large quantities of certain bone marrow elements is needed to supply sufficient quantities of these elements for use in human therapeutics, for example, as bone marrow transplantation, transfusable blood components or genetic therapy.